Development of an Analytical Assay for Electrochemical Detection and Quantification of Protein-Bound 3-Nitrotyrosine in Biological Samples and Comparison with Classical, Antibody-Based Methods.
HPLC with electrochemical detection
mitochondrial superoxide
oxidative stress
peroxynitrite
protein-bound 3-nitrotyrosine
Journal
Antioxidants (Basel, Switzerland)
ISSN: 2076-3921
Titre abrégé: Antioxidants (Basel)
Pays: Switzerland
ID NLM: 101668981
Informations de publication
Date de publication:
06 May 2020
06 May 2020
Historique:
received:
01
04
2020
revised:
30
04
2020
accepted:
02
05
2020
entrez:
10
5
2020
pubmed:
10
5
2020
medline:
10
5
2020
Statut:
epublish
Résumé
Reactive oxygen and nitrogen species (RONS) cause oxidative damage, which is associated with endothelial dysfunction and cardiovascular disease, but may also contribute to redox signaling. Therefore, their precise detection is important for the evaluation of disease mechanisms. Here, we compared three different methods for the detection of 3-nitrotyrosine (3-NT), a marker of nitro-oxidative stress, in biological samples. Nitrated proteins were generated by incubation with peroxynitrite or 3-morpholino sydnonimine (Sin-1) and subjected to total hydrolysis using pronase, a mixture of different proteases. The 3-NT was then separated by high performance liquid chromatography (HPLC) and quantified by electrochemical detection (ECD, CoulArray) and compared to classical methods, namely enzyme-linked immunosorbent assay (ELISA) and dot blot analysis using specific 3-NT antibodies. Calibration curves for authentic 3-NT (detection limit 10 nM) and a concentration-response pattern for 3-NT obtained from digested nitrated bovine serum albumin (BSA) were highly linear over a wide 3-NT concentration range. Also, ex vivo nitration of protein from heart, isolated mitochondria, and serum/plasma could be quantified using the HPLC/ECD method and was confirmed by LC-MS/MS. Of note, nitro-oxidative damage of mitochondria results in increased superoxide (O
Identifiants
pubmed: 32384768
pii: antiox9050388
doi: 10.3390/antiox9050388
pmc: PMC7278855
pii:
doi:
Types de publication
Journal Article
Langues
eng
Subventions
Organisme : Boehringer Ingelheim Stiftung
ID : Novel and neglected cardiovascular risk factors: molecular mechanisms and therapeutics
Organisme : Deutsches Zentrum für Herz-Kreislaufforschung
ID : Partner Site Rhine-Main, Mainz
Déclaration de conflit d'intérêts
Abbreviations: 3-NT, 3-nitrotyrosine; Sin-1, 3-morpholino sydnonimine; PN, peroxynitrite; ROS, reactive oxygen species; Reactive oxygen and nitrogen species (RONS); MnSOD, manganese superoxide dismutase (mitochondrial isoform); HPLC/ECD, high performance liquid chromatography with electrochemical detection; enzyme-linked immunosorbent assay (ELISA) O2•–, superoxide; BSA, bovine serum albumin; mitoSOX, triphenylphosphonium-linked dihydroethidium; NOS, nitric oxide synthase; dTH, dithionite; CoulArray, coulometric array system; retention time (RT).
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